Abstract Sexual dysfunction is a poorly understood condition that affects up to one-third of men around the world. Existing treatments that target the periphery do not work for all men. Previous studies have shown that central melanocortins, which are released by pro-opiomelanocortin neurons in the arcuate nucleus of the hypothalamus, can lead to male erection and increased libido. Several studies specifically implicate the melanocortin 4 receptor (MC4R) in the central control of sexual function, but the specific neural circuitry involved is unknown. We hypothesized that single-minded homolog 1 (Sim1) neurons play an important role in the melanocortin-mediated regulation of male sexual behavior. To test this hypothesis, we examined the sexual behavior of mice expressing MC4R only on Sim1-positive neurons (tbMC4Rsim1 mice) in comparison with tbMC4R null mice and wild-type controls. In tbMC4Rsim1 mice, MC4R reexpression was found in the medial amygdala and paraventricular nucleus of the hypothalamus. These mice were paired with sexually experienced females, and their sexual function and behavior was scored based on mounting, intromission, and ejaculation. tbMC4R null mice showed a longer latency to mount, a reduced intromission efficiency, and an inability to reach ejaculation. Expression of MC4R only on Sim1 neurons reversed the sexual deficits seen in tbMC4R null mice. This study implicates melanocortin signaling via the MC4R on Sim1 neurons in the central control of male sexual behavior. Sexual dysfunction affects a large number of men worldwide. Epidemiological statistics vary depending on the age of the population, the type of sexual dysfunction in question, and the comorbid factors considered (1). Most studies focus on erectile dysfunction, although men can also experience difficulties with interest, desire, ejaculation, and orgasm (1, 2). Between 9% and 54% of men across different countries have been reported to have experienced erectile dysfunction, with incidence increasing with age (3–6). Ejaculation disorders, particularly premature ejaculation, are reported by 20% to 30% of men (7, 8). Treatment options for men experiencing erectile dysfunction include phosphodiesterase 5 inhibitors, such as Viagra, injection of the penile tissue with prostaglandin E1 or similar drugs, and surgical prostheses (9, 10). Phosphodiesterase 5 inhibitors improve erectile function in a substantial number of men, but in some cases, particularly those with underlying conditions such as diabetes, these medications are ineffective (11, 12). For these patients, it is important to consider central mechanisms as therapeutic targets for sexual dysfunction. For premature ejaculation, selective serotonin reuptake inhibitors and local anesthetics have been used for cases that do not seem to have a secondary, treatable cause (9). Another ejaculatory disorder, delayed ejaculation, is less commonly reported and lacks effective treatments (13). The melanocortin system is a promising central target for the treatment of sexual dysfunction in men. One study found that a melanocortin 3/4 receptor agonist, bremelanotide, increased satisfaction levels in men who were also taking Viagra (14). Bremelanotide has also been found to increase blood pressure and decrease heart rate (15). A melanocyte-stimulating hormone (MSH) analog, Melanotan-II, has been found to induce erections in men, but it has a high incidence of unwanted side effects such as nausea and excessive yawning (16, 17). The results of these studies confirm the importance of understanding which central pathways mediate the effects of melanocortins, not only to develop treatments for sexual dysfunction, but also to minimize side effects of pharmacotherapy. Mouse models offer an excellent avenue for understanding the mechanism underlying melanocortin-driven sexual behavior and function. For example, mice lacking the melanocortin 4 receptor (MC4R) are reported to show reduced sexual motivation as well as reduced ejaculation efficiency (18). To better understand the interaction between the central nervous system and sexual function, our laboratory has investigated the involvement of the pro-opiomelanocortin (POMC) system. Mice bred to have POMC neurons that were insensitive to circulating insulin and leptin were found to show decreased mounting behavior, indicating reduced sexual motivation (19). These mice also showed reduced αMSH production and reduced expression of MC4R. Despite rodent and human studies implicating the melanocortins in sexual behavior, the brain nuclei containing the critical melanocortin receptors are unknown. The MC4R, primarily found in the brain, is known to be located in key hypothalamic nuclei downstream of POMC neurons (20). Balthasar and colleagues previously generated a mouse model in which MC4R is expressed in specific tissues that express single-minded homolog 1 (Sim1) (21, 22). Sim1 is a transcription factor expressed in the paraventricular nucleus of the hypothalamus (PVH), basomedial amygdala, anterior hypothalamus, and lateral hypothalamic area (23), regions that also have high concentrations of MCR4-expressing neurons. Using the cre-lox system to specifically express MC4R only on Sim1-cre neurons, it was shown that Sim1 MC4Rs are involved in satiety. tbMC4R null mice become obese over time, but expressing MC4R solely on Sim1 neurons attenuated this phenotype (21). Further studies have found evidence that this effect may be mediated by glutamate (22, 24). In the current study, we use this mouse model as a tool to explore the neurocircuitry of MC4R-mediated sexual behavior. Specifically, we test the specific involvement of Sim1-cre MC4Rs in sexual performance parameters in male mice. Ultimately, understanding the mechanisms underlying melanocortin-mediated sexual behavior may pave the way for future treatments of male sexual dysfunction. Materials and Methods Animal production and care tbMC4R null mice, a previously established mouse model, were purchased from The Jackson Laboratory (loxTB Mc4r; catalog no. 006414). The transcription blocker preventing expression of MC4R in these mice is flanked by loxp sites, such that the presence of cre recombinase will result in the removal of the transcription blocker and subsequent expression of MC4R in tissue-specific sites. Generation of tbMC4Rsim1 mice was accomplished by breeding Sim1-cre mice (The Jackson Laboratory; catalog no. 006395) with mice heterozygous for the tbMC4R null allele. Experimental mice were bred to be hemizygous for Sim1-cre but homozygous for the tbMC4R null allele. To assist with visualization of Sim1 neurons, these mice were also bred with mice expressing a cre-dependent tdTomato reporter (The Jackson Laboratory; catalog no. 007909). Control mice included wild-type (WT) littermates as well as mice that were only hemizygous for Sim1-cre but had normal expression of the MC4R gene. In all studies, tbMC4R null, tbMC4Rsim1, and WT littermate control mice were tested concurrently. Genotyping was confirmed by sending tissue to Transnetyx, Inc., for testing by real-time polymerase chain reaction. Mice were housed in the University of Toledo College of Medicine Department of Laboratory Animal Resources facilities where they were given ad libitum food and water on a 12:12 light:dark cycle with lights off at 6 pm. Food was standard rodent chow (Envigo; catalog no. 2916). All procedures were reviewed and approved by the University of Toledo College of Medicine Animal Care and Use Committee. Sexual behavior Before 6 months of age, males were exposed to primed, ovariectomized female mice to gain sexual experience; males were paired with an experienced female for four separate nights with at least 3 days between pairings. To prime the females, a subcutaneous 100-µL dose of β-estradiol-3-benzoate in sesame oil (200 µg/mL) was given 48 hours before pairing; an intraperitoneal 125-µL dose of progesterone (4 mg/mL) was then given 7 hours before pairing (25, 26). Pairing was done from 8 pm to 9 am during the normal period of activity for mice. After male mice reached 6 months of age, pairing was videotaped (DVR Swann 4500 and T850 Day and Night Security Camera security system) and analyzed for sexual behaviors between 8 pm and 2 am. This timespan was determined to include nearly all relevant sexual behaviors. Red lighting was present when the researchers were setting up the experiment, but was turned off for the duration of filming using night vision cameras. Sexual behaviors measured included anogenital sniffing, mounting, intromission, and ejaculation. Mounting was defined as placing two paws on the back of the female and attempting to thrust. Intromission was defined as deep, successful thrusts. The ratio of successful intromission to mounting attempts was used as a measure of intromission efficiency. Ejaculation was defined as the moment during intromission when males froze, fell over, and subsequently lost interest in the female. The same video-recorded mating sessions were also used to analyze initial sexual interest or motivation to copulate. Sexual motivation can be assessed by measuring a male’s interest in a female in the first 10 or 20 minutes after pairing (19); therefore, we analyzed sexual behavior in the first 20 minutes of pairing. Motivational behaviors included length of time spent in the anogenital sniffing phase, latency to mount, and mounting behavior. Latency to mount within 20 minutes was assigned the maximal value of 20 minutes if mounting was not initiated until after that time. Cannulation A guide cannula (PlasticsOne, 2.3 mm) was surgically implanted into the lateral ventricle using a stereotaxic apparatus. Mice were anesthetized using a ketamine/xylazine mixture and then the cannula was implanted using the following coordinates: anteroposterior, −0.22; mediolateral, +1.13; dorsoventral, −1.95. Mice were given 3 days to recover, were singly housed, and then were tested for a stretching, yawning, and grooming reflex in response to αMSH administration as previously described (27, 28). Administration of αMSH or saline was done using a 5-µL Hamilton syringe attached with microrenathane tubing (BrainTree Scientific) to an internal cannula (PlasticsOne, 1 mm). The stretching, yawning, and grooming reflex testing was done between 10 am and 2 pm. One microliter of 3 µg/µL αMSH or 1 µL of 0.9% saline was administered into the lateral ventricle and mice were returned to their home cage. Ten minutes following injection, mice were observed for 2 hours; grooming, stretching, and yawning behaviors were noted in 15-second intervals. Sexual behavior was also tested immediately following administration of αMSH at 8 pm. Before being paired with an experienced partner, mice were given an intracerebroventricular (ICV) injection of either 1 µL of 1 µg/µL αMSH or 1 µL of 0.9% saline using a Hamilton syringe. All mice were tested in both conditions with 3 days between behavioral testing in randomized order. Immunohistochemistry Upon euthanasia, each mouse was perfused and the brain was obtained. The brain was sectioned in 35- to 40-µm slices and then stored in cryoprotectant until immunohistochemistry was performed. To examine the location of MC4R on Sim1 neurons, brain sections were labeled overnight with rabbit anti-MC4R [1:1000; Abcam; catalog no. ab24233; Research Resource Identifier (RRID): AB_2250589]. Td-Tomato was used as a cre reporter and the fluorescence was visible without the aid of an additional antibody. MC4R was visualized with an Alexa Fluor 488 secondary antibody (1:1000; Donkey antirabbit immunoglobulin G Alexa Fluor 488; Invitrogen; catalog no. A21206; RRID: AB_141708) using a confocal microscope (Leica TCS SP5 Laser Scanning Confocal Microscope). Statistics GraphPad Prism was used for all statistical analysis. All data in figures are represented as mean ± standard error of the mean. One-way analysis of variance (ANOVA) was used to compare more than two groups followed by Fisher least significant difference posttests. For experiments that tested the same mice in both a saline and αMSH condition, a two-way ANOVA was used, followed by Fisher least significant difference posttests to compare within conditions. Statistical significance was defined as P < 0.05. In figure legends, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Results No significant difference was found between WT controls and Sim1-cre controls for weight (P = 0.6099), mounting (P = 0.3402), or any other parameter; therefore, for ease of interpretation, WT mice were used as the control mice in all figures. In all studies, tbMC4R null, tbMC4Rsim1, and WT littermate control mice were tested at the same time. However, to improve the logical flow of the analysis, the results of tbMC4R null and WT mice are presented before the comparison between tbMC4R null and tbMC4Rsim1 mice. tbMC4R null mice have impaired sexual function Sexual function was assessed in 6-month-old male tbMC4R null mice by pairing them with hormonally primed ovariectomized females. These mice exhibited the expected phenotype of increased weight gain (P < 0.0001), which has been well established in the literature (22) (Fig. 1A). Males showed no difference in anogenital sniffing, mounting attempts, latency to mount, or number of times intromission was reached (Fig. 1B–1D). However, mice were found to have a trend toward decreased intromission efficiency (P = 0.0506) (Fig. 1E). Intromission efficiency was defined as the percentage of times intromission was reached during mounting attempts. The ability of the males to reach intromission suggests that they were able to achieve penile erection. However, because these mice required more mounting attempts to reach intromission than controls, these mice may have had difficulty maintaining sufficient penile rigidity. tbMC4R null mice also had a complete inability to achieve ejaculation (P = 0.0009) compared with WT mice (Fig. 1F). These results indicate that tbMC4R null mice have sexual dysfunction that includes a substantially impaired ability to ejaculate. Figure 1. View largeDownload slide Sexual behavior in 6-month-old tbMC4R null and tbMC4Rsim1 mice recorded after pairing between 8 pm and 2 am. (A) Weight gain was significantly different across groups. Posttests revealed tbMC4R null and tbMC4Rsim1 mice had substantial weight gain compared with WT controls (n = 9–12). Time engaged in (B) anogenital sniffing and (C) mounting attempts between WT, tbMC4R null mice, and tbMC4Rsim1 (n = 8–10) was not significantly different. There was also no significance between groups with (D) latency to mount or (E) intromission number. (F) A significant difference was seen between tbMC4R null and tbMC4Rsim1 mice in intromission efficiency. Intromission efficiency showed a trend (P = 0.0506) toward being lower in tbMC4R null mice as well. (G) Percentage to reach ejaculation was significantly higher in both WT and tbMC4Rsim1 mice than tbMC4R nulls. **P < 0.01; ***P < 0.001; ****P < 0.0001. Figure 1. View largeDownload slide Sexual behavior in 6-month-old tbMC4R null and tbMC4Rsim1 mice recorded after pairing between 8 pm and 2 am. (A) Weight gain was significantly different across groups. Posttests revealed tbMC4R null and tbMC4Rsim1 mice had substantial weight gain compared with WT controls (n = 9–12). Time engaged in (B) anogenital sniffing and (C) mounting attempts between WT, tbMC4R null mice, and tbMC4Rsim1 (n = 8–10) was not significantly different. There was also no significance between groups with (D) latency to mount or (E) intromission number. (F) A significant difference was seen between tbMC4R null and tbMC4Rsim1 mice in intromission efficiency. Intromission efficiency showed a trend (P = 0.0506) toward being lower in tbMC4R null mice as well. (G) Percentage to reach ejaculation was significantly higher in both WT and tbMC4Rsim1 mice than tbMC4R nulls. **P < 0.01; ***P < 0.001; ****P < 0.0001. MC4R on Sim1 neurons are important for sexual function Mice were generated to express MC4R solely on Sim1 neurons. Consistent with previous studies, colocalization of MC4R and Sim1 neurons in WT mice was confirmed in the PVH (Fig. 2A and 2B), medial amygdala (MeA) (Fig. 2C), and to a small extent in the supraoptic nucleus of the hypothalamus (SON) (Fig. 2D) (21). In tbMC4Rsim1 mice, colocalization was confirmed in the PVH (Fig. 3A) and MeA (Fig. 3B), but not in the SON (Fig. 3C), possibly because of lower Sim1-cre expression in that location. We confirmed previous studies that found that these mice also have a phenotype of weight gain compared with WT (P = 0.0001) and Sim1-cre (P = 0.0013) control mice (Fig. 1A). However, no weight difference was seen compared with tbMC4R null mice. Figure 2. View largeDownload slide Immunofluorescence showing colocalization of Sim1-cre with MC4R in Sim1-cre-only control mice. Sim1-cre neurons are identified using a Tdtomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. (A) The PVH at ×20 magnification. (B) A zoomed-in picture of the PVH at ×40 magnification shows colocalization more clearly. (C, D) The MeA and SON, which are also Sim1-expressing regions, both show some colocalization with MC4R. Figure 2. View largeDownload slide Immunofluorescence showing colocalization of Sim1-cre with MC4R in Sim1-cre-only control mice. Sim1-cre neurons are identified using a Tdtomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. (A) The PVH at ×20 magnification. (B) A zoomed-in picture of the PVH at ×40 magnification shows colocalization more clearly. (C, D) The MeA and SON, which are also Sim1-expressing regions, both show some colocalization with MC4R. Figure 3. View largeDownload slide Immunofluorescence showing colocalization of Sim1-cre with MC4R in tbMC4Rsim1 mice. Sim1-cre neurons are identified using a Td-tomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. (A) The PVH. (B) The MeA. (C) The SON, which did not show colocalization with MC4R. All images were taken at a ×40 magnification. Figure 3. View largeDownload slide Immunofluorescence showing colocalization of Sim1-cre with MC4R in tbMC4Rsim1 mice. Sim1-cre neurons are identified using a Td-tomato reporter (red), MC4R is visible using a green fluorescent labeled secondary antibody, and the overlay of the two is visible in yellow. (A) The PVH. (B) The MeA. (C) The SON, which did not show colocalization with MC4R. All images were taken at a ×40 magnification. Sexual dysfunction was assessed in 6-month-old male tbMC4Rsim1 mice and then compared with both tbMC4R null mice and controls. The number of mounting attempts was not different between any groups (Fig. 1B). Unlike tbMC4R null mice, tbMC4Rsim1 mice showed normal sexual parameters (Fig. 1B–1F). Intromission efficiency was significantly different across groups [F(2,25) = 4.472, P = 0.0219], with a significant increase in efficiency in tbMC4Rsim1 mice compared with tbMC4R null (P = 0.0073) (Fig. 1E). There was also a significant difference across genotypes in the percentage to reach ejaculation [F(2,25) = 7.331, P = 0.0031] resulting from a recovery of the ability to ejaculate in tbMC4Rsim1 mice compared with tbMC4R null mice (P = 0.0041) (Fig. 1F). This finding suggests that MC4Rs on Sim1 neurons are sufficient to mediate the effects of melanocortins on erectile function and ejaculation. Willingness to mount, as measured by behaviors such as the latency to mount and mounting attempts in the initial pairing period, have been shown to indicate the sexual motivation of the male and has been considered analogous to sexual desire in humans (29). Although tbMC4R null mice exhibited no differences in number of mounts within the initial 20 minutes (Fig. 4A), their latency to mount was significantly increased (P = 0.0472) (Fig. 4B). This finding is consistent with previous studies that examined the effect of MC4R on sexual behavior in mice (18). In tbMC4Rsim1 mice, sexual motivation did not differ statistically from controls (Fig. 4A and 4B), suggesting that the motivational defects seen in the tbMC4R null are mediated at least partly by Sim1 neurons. Figure 4. View largeDownload slide Sexual motivation was partially impaired by knocking out MC4R. (A) Mounting attempts of WT, tbMC4Rsim1, and tbMC4R null mice (n = 8–10) in the first 20 minutes of the test were not significantly different by one-way ANOVA. (B) However, a substantial increase was seen in the latency to mount in tbMC4R null mice. *P < 0.05. Figure 4. View largeDownload slide Sexual motivation was partially impaired by knocking out MC4R. (A) Mounting attempts of WT, tbMC4Rsim1, and tbMC4R null mice (n = 8–10) in the first 20 minutes of the test were not significantly different by one-way ANOVA. (B) However, a substantial increase was seen in the latency to mount in tbMC4R null mice. *P < 0.05. Exogenous αMSH affects grooming behavior through Sim1 neurons To assess the effect of exogenous αMSH on sexual function, sexual behavior was tested after ICV administration of 1 µg of αMSH. Interestingly, αMSH had no effect on anogenital sniffing, number of mounts, latency to mount, intromission efficiency, or ability to reach ejaculation in WT mice (not shown). These results precluded further study in MC4RKO and tbMC4Rsim1 mice. Effects on other αMSH-induced behaviors were measurable in response to ICV peptide administration. Exogenous αMSH has been reported to induce a stretching-yawning-grooming response, which is also accompanied by penile erection in rats (27, 30). We and others have found that it is not feasible to visualize spontaneous erections in behaving mice (18, 19). Thus, we investigated whether ICV administration of αMSH works through PVH MC4R to elicit behavioral responses. αMSH did significantly increase grooming (P = 0.0002), stretching (P = 0.0003), and yawning (P = 0.0172) behaviors in control animals (Fig. 5A–5C). We found that tbMC4R null mice did not have increased grooming, stretching, or yawning in response to αMSH administration, but only grooming was found to be induced by αMSH in tbMC4Rsim1 mice (P = 0.0411) (Fig. 5A). There was a significant interaction [F(2, 54) = 6.293, P = 0.0035], significant effect of genotype [F(2, 54) = 4.419, P = 0.0167], and significant effect of treatment [F(1,54) = 8.861, P = 0.0044] for grooming behaviors. A two-way ANOVA on stretching behavior also revealed a significant interaction [F(2, 54) = 3.622, P = 0.0334] with a main effect of genotype [F(2, 54) = 6.637, P = 0.0026] and treatment [F(1, 54) = 9.000, P = 0.0041]. The main effect of genotype shows that WT mice stretched more often than either tbMC4R null (P = 0.0009) or tbMC4Rsim1 mice (P = 0.0126). There was no substantial interaction or main effects on yawning behavior. These data suggest that only the grooming response to central αMSH administration is mediated by MC4R on Sim1 neurons. Figure 5. View largeDownload slide Grooming induced by ICV αMSH requires MC4Rs on Sim1 neurons. Behaviors were observed between 10 and 130 minutes postinjection. (A) ICV administration of αMSH significantly increased time spent grooming in both the WT and tbMC4Rsim1 groups. (B) Stretching behavior was also significantly increased in WT mice after αMSH administration, but not in tbMC4R null or tbMC4Rsim1 mice. (C) Similarly, αMSH administration increased only yawning in WT mice. n = 8–11 in all groups. *P < 0.05; ***P < 0.001. Figure 5. View largeDownload slide Grooming induced by ICV αMSH requires MC4Rs on Sim1 neurons. Behaviors were observed between 10 and 130 minutes postinjection. (A) ICV administration of αMSH significantly increased time spent grooming in both the WT and tbMC4Rsim1 groups. (B) Stretching behavior was also significantly increased in WT mice after αMSH administration, but not in tbMC4R null or tbMC4Rsim1 mice. (C) Similarly, αMSH administration increased only yawning in WT mice. n = 8–11 in all groups. *P < 0.05; ***P < 0.001. Discussion In this study, we found that tbMC4R null mice have a complete inability to reach ejaculation as well as decreased intromission efficiency. These parameters were restored by expressing MC4R only on Sim1 neurons, indicating that these receptors in Sim1 neurons are sufficient to permit normal erectile function and ejaculation. This study demonstrates that direct melanocortin action on Sim1 target neurons is necessary for sexual function. Taking into account our previous studies in mice with leptin and insulin-insensitive POMC neurons (19), this study provides evidence that MC4R-expressing Sim1 neurons receive input from arcuate POMC neurons and together form part of a neural circuit underlying male sexual function. We tested several aspects of the sexual response using a behavioral paradigm that allowed us to assess the effect of melanocortin signaling on sexual interest, erectile function, and ejaculation. Sexual motivation, the human analog of sexual desire, was investigated via precopulatory behaviors (anogenital sniffing) and willingness to engage in mounting behavior (latency to mount and initial mounting attempts) (29). We saw an increase in latency to mount in tbMC4R null mice, but no other difference in sexual motivation. In contrast, Van der Ploeg and others (18, 19) found a lack of sexual motivation in tbMC4R null mice that was not seen in our studies. Unlike that report, we ensured that all mice in our study had previous copulatory experience before testing. This step may have eliminated delays in engaging in sexual activity resulting from inexperience and minimized differences between groups. We hypothesized that exogenous αMSH would result in improved sexual function in control mice and tbMC4Rsim1 mice. Surprisingly, ICV αMSH had no effect on copulatory function control animals. This finding may indicate that the normal copulatory function of control mice could not be further improved by αMSH because of a physiological ceiling. Alternatively, improvement may be possible in control animals under other experimental paradigms; specifically, previous pharmacological studies primarily investigated noncontact erection, whereas we measured copulatory function. Indeed, contact erection may rely on different mechanisms compared with noncontact erection (31). This pharmacological experiment adds to wealth of complex data regarding the effect of centrally administered αMSH on copulatory function. The percent of mice able to achieve ejaculation was also examined. We found that tbMC4R null mice were unable to reach ejaculation, whereas that ability was restored in tbMC4Rsim1 mice. These findings are consistent with a previous study showing reduced ejaculation efficiency in MC4RKO mice (18). These data support a role for Sim1-expressing neurons such as those in the PVH in ejaculatory function, as previously suggested by findings of reduced intromission efficiency and increased ejaculation latencies in rats with PVH lesions (32). In mice, as in humans, ejaculation is a clear, measurable response. Ejaculation is heavily controlled by autonomic and motor neurons through the spinal cord. Lesion and tracing studies in animals have implicated brain regions such as the medial preoptic area as well as the PVH in the central control of ejaculation (33). One study using positron emission tomography to examine brain activity in humans during ejaculation found activity in many mesodiencephalic regions, but not in the hypothalamus (34). Although it has been suggested that rodents may experience orgasm-like responses (35), we were unable to measure orgasm in our mice. Interestingly, it has been suggested that the loss of interest felt following ejaculation is similar to the sensation of satiety felt after eating (36), a state in which the MC4R is known to be involved. Studies have found that administering MC4R agonists into rodents’ lateral ventricles of the brain results in erection, along with yawning, stretching, and grooming (27, 37). Although it is feasible to observe the penis emerge from the penile sheath in rats, we and others have found that this approach is not feasible in mice because of their smaller size (18, 19). Nevertheless, other actions of central MC4R agonists are readily observed. We hypothesized that ICV administration of αMSH would increase these melanocortin-mediated behaviors in all mice except tbMC4R null mice. Although we did see increases in grooming and stretching induced by ICV αMSH in control animals, only grooming was increased in tbMC4Rsim1 mice. This effect may indicate that MC4Rs in Sim1 neurons regulate grooming, but not stretching and yawning. This result is supported by findings that αMSH in the paraventricular nucleus elicits a grooming response (38, 39). Interestingly, one study noted that these αMSH-induced behaviors only occur in the presence of stressful handling procedures. These authors suggest that αMSH maintains grooming but does not initiate it, so these behaviors might not be present in the absence of stress (40). Understanding the neurocircuitry underlying these behaviors may permit targeted drug development for erectile dysfunction without such unwanted side effects. MC4R on Sim1 neurons also play a role in regulating eating behavior (21, 22, 24). Obesity is thought to contribute to sexual dysfunction in men (41). As seen in previous studies, the tbMC4R null mice did become considerably obese (21, 22). Our tbMC4Rsim1 mice were also obese, which is supported by the literature, although at this age we did not see the reportedly attenuated weight gain compared with tbMC4R null mice at 12 weeks of age (21). Notably, a previous report shows the difference in body weight between tbMC4Rsim1 and tbMC4R mice lessening as the mice approach 5 months of age (24). It is unknown how obesity affected the results of these studies, although it is clear that tbMC4Rsim1 mice were able to mate as well as controls despite marked obesity. Additional studies will be required to disentangle the effects of obesity and melanocortin deficiency. The brain circuitry controlling sexual function is complex. In rodents, prerequisite olfactory cues converge on the MeA, bed nucleus of the stria terminalis, and hypothalamic nuclei (42–45). These areas are interconnected and essential to the control of mating and aggression behaviors in both sexes (46–52). GABAergic MeA neurons mediate both male mating and aggression (53). Neurons in the MeA, in turn, project to targets including the lateral septum, hypothalamus, and bed nucleus of the stria terminalis. Hypothalamic areas traditionally known to play a role in sexual behaviors include the medial preoptic area and its subnucleus, the medial preoptic nucleus, and the ventromedial nucleus (54–58). In humans, functional magnetic resonance imaging studies have also implicated the amygdala and hypothalamus in regulating erection (59). Both the medial preoptic area and ventromedial nucleus project to lateral regions of periaqueductal gray that integrate motor and autonomic inputs of sexual behavior (60, 61). More investigation is necessary to determine which Sim1 neurons underlie the restoration of successful intromission and ejaculation in tbMC4Rsim1 mice. Sim1 expression has been reported in the PVH, posterior hypothalamic area, supraoptic nucleus, periaqueductal gray, nucleus of the lateral olfactory tract, MeA, and preoptic anterior hypothalamic nucleus (62). Of these areas, MC4R expression in the mouse has been reported in the PVH, SON, anterior hypothalamic nucleus, and MeA (63). We confirmed MC4R reexpression in the PVH and MeA, but did not see any in the anterior hypothalamic nucleus or SON. The amygdala is a prime candidate for playing a role in melanocortin-dependent sexual function. Stimulation of the amygdala will produce penile erection, sexual sensation, representations/memories of intercourse, and orgasm in humans (64–66). Lesions of the corticomedial amygdala produce sexual behavior deficits in male rats (67–71). Male hamsters with lesions in the anterior dorsal part of the MeA failed to engage in any chemoinvestigatory or copulatory activities, whereas those with lesions of the posterior dorsal part of the MeA showed a modest decrease in chemoinvestigation and a greater latency to ejaculation (72–74). As mentioned, the MeA expresses both Sim1 and the MC4R; therefore, restoration of MC4Rs in this region may underlie the normalized phenotype of tbMC4R sim1 mice. It is also possible that the PVH plays a role in the observed effects. A tracing study that injected pseudorabies virus into the corpus cavernosus of rats found virus in the paraventricular nucleus (75). Furthermore, a study in rats found that lesioned parvocellular and magnocellular neurons in the paraventricular nucleus resulted in delayed ejaculation and reduced intromission ratios (32), supporting a role for PVH neurons in sexual function. Paraventricular nucleus projections to serotonergic neurons in the medulla have been found to be involved in regulating male sexual function (76). Sim1-expressing cells in that nucleus include oxytocin, thyrotropin-releasing hormone, corticotropin-releasing hormone, vasopressin, and somatostatin neurons (77); both oxytocin and serotonin neuronal projections have been found on preganglionic sacral neurons that control penile function (78). Narrowing down the relevant neurocircuitry from among the possible sites of MC4R-expressing Sim1 neurons may assist in the development of drugs that can target sexual function without affecting any other melanocortin pathways. Finally, it is important to recognize that a comprehensive determination of the targets for melanocortin-mediated sexual behavior remains to be done. The present studies have demonstrated that MC4R expression on Sim1 neurons is sufficient to mediate melanocortin-dependent sexual function in behaving mice. However, other targets may serve a similar function. Additional work is needed to test whether Sim1 melanocortin sensing is necessary for male sexual function. In conclusion, we have shown that MC4R signaling in Sim1 neurons regulates the ability to achieve intromission and ejaculation in male mice at 6 months of age. These receptors also mediate grooming behavior, but not stretching or yawning. These results and future investigation of implicated melanocortin neurocircuitry may allow for the development of more specific therapeutic targets for improving sexual function. In addition, determining how these melanocortin circuits diverge from those regulating energy balance and metabolism may permit the development of weight loss medications free of sexual side effects. Appendix. Antibody Table Peptide/Protein Target Antigen Sequence (if Known) Name of Antibody Manufacturer, Catalog No. Species Raised in; Monoclonal or Polyclonal Dilution Used RRID MC4R Rabbit anti-MC4R Abcam, ab24233 Rabbit; polyclonal 1:1000 AB_141708 Rabbit IgG Donkey anti-rabbit IgG Alexa Fluor 488 Invitrogen, A21206 Donkey; polyclonal 1:1000 AB_2250589 Peptide/Protein Target Antigen Sequence (if Known) Name of Antibody Manufacturer, Catalog No. Species Raised in; Monoclonal or Polyclonal Dilution Used RRID MC4R Rabbit anti-MC4R Abcam, ab24233 Rabbit; polyclonal 1:1000 AB_141708 Rabbit IgG Donkey anti-rabbit IgG Alexa Fluor 488 Invitrogen, A21206 Donkey; polyclonal 1:1000 AB_2250589 View Large Abbreviations: ICV intracerebroventricular MC4R melanocortin 4 receptor MeA medial amygdala MSH melanocyte-stimulating hormone POMC pro-opiomelanocortin PVH paraventricular nucleus of the hypothalamus RRID Research Resource Identifier Sim1 single-minded homolog 1 SON supraoptic nucleus of the hypothalamus WT wild-type. 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